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1 <tool id="htseqsams2mxlocal" name="SAM/BAM to count matrix" version="0.4">
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2 <description>using HTSeq code</description>
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3 <stdio>
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4 <regex match=".*" source="both" level="warning" description="chatter from HTSeq:"/>
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5 </stdio>
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6 <requirements>
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7 <requirement type="package" version="0.7.6">pysam</requirement>
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8 <requirement type="package" version="1.2">matplotlib</requirement>
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9 <requirement type="package" version="0.5.4p3">htseq</requirement>
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10 </requirements>
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11 <command interpreter="python">
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12 htseqsams2mx.py -g "$gfffile" -o "$outfile" -m "$model" --id_attribute "$id_attr" --feature_type "$feature_type"
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13 --mapqMin $mapqMin --samf "'${firstsamf}','${firstsamf.name}','${firstsamf.ext}','${firstsamf.metadata.bam_index}'"
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14 #if $secondsamf.ext != 'data':
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15 --samf "'${secondsamf}','${secondsamf.name}','${secondsamf.ext}','${secondsamf.metadata.bam_index}'"
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16 #end if
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17 #for $s in $samfiles:
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18 #if $s.samf.ext != 'data':
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19 --samf "'${s.samf}','${s.samf.name}','${s.samf.ext}','${s.samf.metadata.bam_index}'"
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20 #end if
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21 #end for
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22 #if $filter_extras:
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23 --filter_extras "$filter_extras"
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24 #end if
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25 </command>
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26 <inputs>
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27 <param format="gtf" name="gfffile" type="data" label="Gene model (GFF) file to count reads over from your current history" size="100" />
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28 <param name="mapqMin" label="Filter reads with mapq below than this value"
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29 help="0 to count any mapping quality read. Otherwise only reads at or above specified mapq will be counted"
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30 type="integer" value="5"/>
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31 <param name="title" label="Name for this job's output file" type="text" size="80" value="bams to DGE count matrix"/>
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32 <param name="stranded" value="false" type="boolean" label="Reads are stranded - use strand in counting" display="checkbox"
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33 truevalue="yes" falsevalue="no" checked="no" help="Check this ONLY if you know your sequences are strand specific" />
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34 <param name="model" type="select" label="Model for counting reads over the supplied gene model- see HTSeq docs"
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35 help="If in doubt, union is a reasonable default but intersection-strict avoids double counting over overlapping exons">
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36 <option value="union" selected="true">union</option>
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37 <option value="intersection-strict">intersection-strict</option>
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38 <option value="intersection-nonempty">intersection-nonempty</option>
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39 </param>
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40 <param name="id_attr" type="select" label="GTF attribute to output as the name for each contig - see HTSeq docs"
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41 help="If in doubt, use gene name or if you need the id in your GTF, gene id">
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42 <option value="gene_name" selected="true">gene name</option>
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43 <option value="gene_id">gene id</option>
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44 <option value="transcript_id">transcript id</option>
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45 <option value="transcript_name">transcript name</option>
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46 </param>
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47 <param name="feature_type" type="select" label="GTF feature type for counting reads over the supplied gene model- see HTSeq docs"
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48 help="GTF feature type to count over - exon is a good choice with gene name as the contig to count over">
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49 <option value="exon" selected="true">exon</option>
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50 <option value="CDS">CDS</option>
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51 <option value="UTR">UTR</option>
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52 <option value="transcript">transcript</option>
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53 </param>
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54 <param name="filter_extras" type="select" label="Filter any read with one or more flags"
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55 help="eg the XS tag created by bowtie for multiple reads" optional="true" mutliple="true">
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56 <option value="">None</option>
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57 <option value="XS">XS:i > 0 - More than one mapping position Bowtie</option>
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58 <option value="XS:A">Might be useful for tophat</option>
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59 </param>
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60
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61 <param name="firstsamf" type="data" label="bam/sam file from your history" format="sam,bam" size="100"
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62 help="Each sam/bam contributes a column of read counts overlapping the specified gene model contigs"
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63 optional="false"/>
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64 <param name="secondsamf" type="data" label="Additional bam/sam file from your history" format="sam,bam" size="100"
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65 help="Each sam/bam contributes a column of read counts overlapping the specified gene model contigs"
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66 optional="false"/>
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67 <repeat name="samfiles" min="16"
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68 title="Specify additional bam/sam file inputs" help="Each sam/bam contributes a column of read counts overlapping the specified gene model contigs">
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69 <param name="samf" type="data" label="Additional bam/sam file from your history" format="sam,bam" size="100"
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70 optional="true"/>
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71 </repeat>
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72 </inputs>
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73 <outputs>
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74 <data format="tabular" name="outfile" label="${title}_htseqsams2mx.xls" />
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75 </outputs>
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76 <tests>
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77 <test>
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78 <param name="feature_type" value="exon" />
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79 <param name="gfffile" value="rn4_chr20_100k.gtf" />
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80 <param name="firstsamf" value="rn4chr20test1.bam" ftype="bam"/>
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81 <param name="secondsamf" value="rn4chr20test2.bam" ftype="bam"/>
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82 <param name="id_attr" value="gene_name" />
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83 <param name="model" value="union" />
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84 <param name="stranded" value="no" />
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85 <param name="title" value="htseqtest" />
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86 <param name="mapqMin" value="0" />
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87
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88 <output name="outfile" file="htseqsams2mx_test1_out.xls" lines_diff="1"/>
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89 </test>
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90 </tests>
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91 <help>
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92
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93 **What this tool does**
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94
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95 Counts reads in multiple sam/bam format mapped files and generates a matrix ideal for edgeR and other count based tools
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96 It uses HTSeq to count your sam reads over a gene model supplied as a GTF file
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97 The output is a tabular text (columnar - spreadsheet) file containing the
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98 count matrix for downstream processing. Each row contains the counts from each sample for each
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99 of the non-emtpy GTF input file contigs matching the GTF attribute choice above.
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100 You probably want to use gene level GTF output attribute and count reads that overlap
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101 GTF exons for RNA-seq. Or you can count over exons by using transcript level output names or ids. Etc.
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102
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103 ----
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104
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105 **Author's plea on replicates**
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106
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107 If you want to interpret the downstream p values in terms of rejecting or accepting the null hypothesis
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108 under random sampling with replacement from the universe of possible biological/experimental replicates from which your data was derived,
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109 which is what published p values are often assumed to do, then you need biological
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110 (or for cell culture material experimental) replicates.
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111
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112 Using technical or no replicates means the downstream p values are not interpretable the way most people would assume
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113 they are - ie as the probability of obtaining a result as or more extreme as your experimental data
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114 in millions of experiments conducted using the same methods under the null hypothesis.
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115
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116 There is no way around this and it is scientific fraud to ignore this issue and publish bogus p values derived from
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117 technical or no replicates without making the lack of biological or experimental error in the p value calculations
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118 clear to your readers so they can adjust their expectations. However, the buck stops here at higher level inference.
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119 If you have no replicates, you must not use this tool as the p values are uninterpretable. So there.
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120
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121 See your stats 101 notes on the central limit theorem and test statistics for a refresher or talk to a
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122 statistician if this makes no sense please.
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123
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124 **Attribution**
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125
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126 This Galaxy tool relies on HTSeq_ from http://www-huber.embl.de/users/anders/HTSeq/doc/index.html
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127 for the tricky work of counting. That code includes the following attribution:
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128
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129 ## Written by Simon Anders (sanders@fs.tum.de), European Molecular Biology
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130 ## Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General
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131 ## Public License v3. Part of the 'HTSeq' framework, version HTSeq-0.5.4p3
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132
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133 It will be automatically installed if you use the toolshed as in general, you probably should.
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134 HTSeq_ must be installed with this tool if you install manually.
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135
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136 Otherwise, all code and documentation comprising this tool including the requirement
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137 for more than one sample bam
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138 was written by Ross Lazarus and is
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139 licensed to you under the LGPL_ like other rgenetics artefacts
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140
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141 Sorry, I don't use readgroups so had no reason to code read groups. Contributions welcome. Send code
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142
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143 .. _LGPL: http://www.gnu.org/copyleft/lesser.html
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144 .. _HTSeq: http://www-huber.embl.de/users/anders/HTSeq/doc/index.html
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145 </help>
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146
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147 </tool>
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